Antioxidant supplementation to medium for in vitro embryo production in Felis catus

DOI 10.24425/pjvs.2019.129966

Original article

Correspondence to: F. Ciani, e-mail: ciani@unina.it

# _{These authors contributed equally to this work.}

Antioxidant supplementation to medium

for in vitro embryo production in Felis catus

N. Cocchia

_{, S. Tafuri}

_{, C. Del Prete, V. Palumbo, L. Esposito, L. Avallone,}

F. Ciani

Department of Veterinary Medicine and Animal Production

University of Naples Federico II, Via Federico Delpino, 1, 80137 Naples, Italy

Abstract

The development of in vitro embryo production (IVEP) techniques in Felis catus is a fitting model with potential application to the conservation of endangered felid species. To improve the quality of IVEP techniques an appropriate balance of pro- and antioxidants should be provided. Under in vitro conditions, high levels of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) mRNA provide a defence mechanism against oxidative stress for embryos. In order to improve the development of cat oocytes, the effects of SOD and CAT supplemented to in vitro maturation (IVM) medium and of GPx supplemented to in vitro fertilization (IVF) medium on development and embryo production in vitro were evaluated. Data showed an increase of 70 and 77 % of cleaved embryo and blastocyst formation, respec- tively, in the experiment with SOD and CAT addition to IVM medium; in the experiment with GPx addition to IVF medium the number of cleaved embryos doubled and the number of embryos increased by 96 %. Therefore, our results were positive and encourage us to continue studies on cat oocytes evaluating the effects of various dosages and combination of antioxidants.

Key words:

Introduction

In vitro embryo production (IVEP) is an emerging and cutting-edge assisted reproductive technology (ART) that can potentially be applied for the conserva-tion of threatened species in danger of extincconserva-tion. Felis catus represents a practical model for the development of IVEP technology in feline species. Accordingly, the potentially beneficial effects generated by the use of pro- and antioxidants in IVEP are of utmost rele-vance (Cocchia et al. 2010a, 2010b).

Oxidative stress (OS) in spermatozoa strongly affects the success of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) as well as arti-ficial insemination (AI) (Agarwal et al. 2006). A fine balance between pro- and antioxidant species is requi- red for maintaining spermatozoa functions both in male and female genital tracts. Higher concentrations of sper- matozoa are used for IVF than in natural conditions; therefore, the balance between pro- and antioxidant factors requires further evaluation. The determination of pro- and antioxidants in follicular and epididymal

fluids is helpful to better understand the modification in vivo and to optimize in vitro maturation (IVM) envi-ronment for oocytes (Noblac et al. 2011). Under in vitro conditions, the embryo is exposed to potentially damag-ing of internal and external sources of reactive oxygen species (ROS). Because of the nature of standard in vitro culture conditions, the risk of exposure to ROS and OS is much higher than that under in vivo condi-tions (Cocchia et al. 2015a).

During cell metabolism ROS are customary synthe-sized; normal ROS production is necessary for mainte-nance of body functions, while excessive production is harmful (Tafuri et al. 2015). The resulting free radi-cals produce structural and functional alterations by molecular oxidation (Ciani et al. 2018). ROS are counteracted by a complex defense system of enzy-matic and non-enzyenzy-matic antioxidants (Costantino et al. 2009, Ciani et al. 2015, Esposito et al. 2017). Among the enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are the most important ones of the endogenous antioxidant barrier. They are capable to inactivate the excess concentration of radicals like superoxide anion first, and hydroperox-ide after. Enzymatic antioxidants were found in male and female genital tract secretions, including SOD, CAT and GPx (Ciani et al. 2015, Tafuri et al. 2015). SOD, present naturally in seminal plasma, catalyzes the breakdown of the superoxide anion into hydrogen peroxide (Cocchia et al. 2011, Ciani et al. 2015). SOD has been reported to increase the proportion of zygotes that undergo the first cleavage division while improving cleavage past the two-cell stage and blasto-cyst development (Ochota et al. 2016). Elevated levels of SOD may provide protection from gynecologic diseases, such as pre-eclampsia or diabetes-induced embryopa-thy (Agarwal and Allamaneni 2004). CAT is another enzymatic antioxidant present in seminal plasma, in the corpus luteum and in oviduct fluid that can neu-tralize hydrogen peroxide by conversion into oxygen and water (Chi et al. 2008). GPx is present in the mam-malian epididymis and in semen and catalyzes the reduction of hydrogen peroxide and organic perox-ides in water and alcohol while oxidizing glutathione (Galecka et al. 2008, Pipolo et al. 2018). GPx is also located within the glandular epithelium of the uterine endometrium and in the cumulus cells of females and in the sperm mitochondrial matrix and in seminal plasma, so it can come from the prostate (Formigari et al. 2007, Tafuri et al. 2015).

The in vitro model of the ART does not exactly reflect the in vivo conditions of oviducts, but has a greater concentration of radicals that must be counteracted in order to avoid their deleterious action on biological macromolecules and therefore the embryo production

and its survival. The aim of our research, therefore, was to test the effects of supplementation of the oocyte IVM medium with SOD and CAT and the IVF medium with GPx on in vitro developmental competence and embryo production rate in Felis catus.

Materials and Methods

Chemicals and experimental design

Chemical reagents were acquired from Sigma- -Aldrich (Milan, Italy) unless specified different. For the present study ovaries at various phases of the estrous cycle from domestic cats (Felis catus, 1-8 years old) were obtained after ovariohysterectomies carried out in the Veterinary Hospital (OVUD), follow-ing the procedures accordfollow-ing to the Ethical Committee of our University. To determine the efficacy of antioxi-dant supplementation in IVM and IVF medium experi-ments were performed.

To assess the frequency of maturation of oocytes and blastocyst formation, SOD and CAT were added to IVM medium (metaphase II = MII and polar body formation) and the aceto-orcein staining was carried out to estimate the maturation phase of the oocytes. Oocytes were stained in a drop of lacto-aceto-orcein dye for 15 min, and washed in 45 % acetic acid. The stained oocytes were covered with a cover glass to get squash preparations by tapping on the cover glass. The squash preparation were examined under light microscope.

To evaluate the frequency of the maturation of oocytes, cumulus-oocyte complexes (COCs; n=315) were collected and splitted randomly into the experi-mental (n=156) or control (n=159) groups. COCs were cultured as previously described (Pope 2014, Cocchia et al. 2015b). Briefly, IVM was performed using maturation medium (SOFaaBSA) with or without SOD (25 IU/mL) and CAT (50 IU/mL) for 24 h at 38.5°C in 5% CO₂. Then, to determine the chromatin status, COCs were stained with aceto-orcein; only oocytes with an evident metaphase and polar body were classed like meiotically mature (MII).

To evaluate the blastocyst formation, COCs (n = 489) were collected from fresh excised ovaries. The COCs were splitted randomly into experimental (n=243) and control (n=246) groups and grown in maturation medium (SOFaaBSA) supplemented with or without SOD (25 IU/mL) and CAT (50 IU/mL). Then, oocytes were fertilized in vitro in SOFaaBSA in CO₂ (5%) for 18 h at 38.5°C with fresh epididymal spermatozoa. Following IVF, zygotes were cultured in SOFaaBSA with different molecules in CO₂ (5%), N₂ (5%) and O₂ (90%) for 8 days at 38.5°C.

In order to evaluate the efficacy of GPx supplemen-tation in IVF medium, COCs (n=576) were harvested from fresh ovaries and grown for 24 h in maturation medium (SOFaaBSA). They were then splitted into experimental (COCs n=303) and control (COCs n=273) groups and fertilized in vitro in SOFaaBSA medium with or without GPx (25 IU/mL) with epididymal fresh spermatozoa. After 18 h, the zygotes were grown with different molecules in SOFaaBSA. The percentage of oocytes that underwent cleavage/total number of oocytes that underwent IVF x 100 represents the value of cleavage rate. For comparative purposes, from both total number of IVF COCs and cleaved embryos, the blastocyst rate on day 8 was calculated. In vitro development competence was determined as the number of blastocysts produced compared to the total number of embryos.

Oocyte collection and COCs IVM

The experimental procedure was described by Cocchia (2010a). Briefly, the ovaries were stored

in Dulbecco’s PBS with kanamycin (75 mg/mL) until oocyte harvesting at room temperature (RT). Within 3 h of excision, ovaries were chopped with a scalpel in a Petri dish with HEPES synthetic oviductal fluid (HSOF). Oocytes (grade I and II) were selected, washed in HSOF and splitted into the experimental groups (Luvoni 2006).

COCs (25-50 oocytes/mL) were cultured in SOFaaBSA supplemented with amino acids and BSA (6 mg/mL) with porcine follicle-stimulating and luteinizing hormones (0.1 IU; pFSH-LH; Pluset, Laboratorios Calier, Barcelona, Spain); EGF (25 ng/mL); insulin- -transferrin-sodium selenite (25 μL/mL) and L-cysteine (1.2 mmol/L) in CO₂ (5%) at 38.5°C. The oocytes were evaluated with a stereomicroscope after 24 h to assess the viability to remove those with cytoplasmic degener-ation.

Evaluation of the nuclear stage of maturation

After IVM oocytes, denuded in hyaluronidase (0.2% w/v) and placed in KCl solution (0.7% w/v)

Fig. 1. Oocytes stained with aceto-orcein to visualize the nuclear structures (400X magnification). Nuclear morphology was classified as germinal vesicle intact (GV; a), germinal vesicle breakdown (GVBD; b), prometaphase I (PM; c), metaphase (d).

for 3-5 min at RT, were placed on a microslide and fixed overnight in fixative solution (acetic acid/ethanol, 1/3, w/v) (Hewitt et al. 1998). Then, nuclear structures were visualized with aceto-orcein (2% orcein, 45% ace-tic acid) staining and using phase contrast microscopy (400X). Nuclear morphology was assorted as follows: germinal vesicle intact (GV), germinal vesicle break-down (GVBD), metaphase I (MI), MII and undeter-mined nuclear status.

Sperm collection, IVF and IVC

The experimental procedure was described by Cocchia (2015b). Briefly, matured COCs were subjected to IVF with fresh spermatozoa collected from epididymis of domestic cats after orchiectomy at the OVUD. The testicular-epididymal tissue, within 3 h of removal, was placed in Dulbecco’s PBS and carried to our labo-ratory. The caudal portion of each epididymis was minced with a scalpel to release the spermatozoa. Following incubation, the epididymal tissue was elimi-nated, the medium collected and centrifuged. The pellet concentration was assessed by phase-contrast micro- scopy and resuspended in fresh IVF medium to a con-centration of 10 x 106 _{sperm/mL. After incubation,} mo-tile sperms were chosen, the supernatant was collected and supplemented with penicillamine-hypotaurine- -epinephrine (20 mg/mL) and heparin (10 mg/mL).

For IVF, COCs (30-40) were placed in IVF medium and cultured with sperm, then cumulus cells were removed. The denuded oocytes were observed and degenerated ones were discarded. The zygotes were grown in SOFaaBSA (16 mg/mL); cleavage was estab-lished after 24 h of in vitro culture (IVC), and embryos were cultured in medium with fetal bovine serum (10%) until day 8, every 4 days the medium was renovated (Ciani et al. 2008).

Statistical analysis

All data, expressed as mean ± standard deviation (SD), were analyzed with JMP 8.0.2, SAS Institute Inc., USA. Normality was tested with a Shapiro-Wilk’s W test. As not all data were normally distributed, non-parametrical tests were used. The Kruskal-Wallis one-way analysis of variance by rank was applied to compare developmental data in experimental and control groups. Statistical significance was fixed at p≤0.05.

Results

Evaluation of the effect of SOD and CAT supplementation in IVM medium on oocytes

maturation

From each domestic cat 20-30 COCs were collec- ted, of which only those identified as grade I and II COCs were tested. The efficacy of antioxidants supple-mentation in IVM medium was assessed by aceto-orcein staining (Fig. 1). To visualize the nuclear structures aceto-orcein staining was used. Nuclear morphology was identified as follows: germinal vesicle intact (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), MII and undetermined nuclear status.

The results of the effect of SOD and CAT supple-mentation to IVM medium in each group are shown in Table 1. SOD and CAT in IVM medium were not effective in improving oocyte maturation, in fact matu-ration rate did not show any significant difference between control and experimental groups (Table 1).

Table 1. Evaluation of the effect of SOD and CAT supplementation to in vitro maturation (IVM) medium on oocytes maturation. N. COCs IVM

(mean ± SD) (mean ± SD) (%)MII

Control group 16.24±3.42 13.68 ± 2.13 (81)

Experimental group 18.27±1.43 13.76 ±3.54 (79)

COCs: cumulus-oocyte complexes

Table 2. Evaluation of the efficacy of SOD and CAT supplementation to in vitro maturation (IVM) medium on embryo cleavage and blastocyst formation.

N. COCs IVC

(mean ± SD) (mean ± SD) (%)Cleaved embryo (mean ± SD) (%)N. Blastocyst

Control group 18.15±2.11 6.34± 2.68 (33) *A 3.18 ±0.65 (16) *A

Experimental group 16.88±3.26 11.25±3.08 (65) *B 5.41±1.04 (32) *B

* p < 0.05 A vs B columns COCs: cumulus-oocyte complexes; IVC: in vitro culture

Evaluation of the efficacy of SOD and CAT supplementation in IVM medium on embryo

cleavage and blastocyst formation

From each domestic cat 20-30 COCs were collec- ted, of which only those classified as grade I and II were tested. The results of SOD and CAT supplementation on IVM medium in each group are reported in Table 2. The frequencies of cleavage and blastocyst develop-ment were greater in experidevelop-mental group in respect to the control (p<0.05).

Evaluation of the efficacy of GPx supplementation in IVF medium on embryo cleavage and blastocyst

formation

From each domestic cat 20-35 COCs were collec- ted, of which only those classified as grade I and II COCs were tested. In this set of experiments develop-mental competence, as number of blastocysts producted and embryos, was evaluated. The efficacy of GPx sup-plementation to IVF medium in each group are shown in Table 3. Data showed that the frequency of cleavage was significantly increased in GPx supplementation group with respect to control group (p<0.05). More-over, blastocyst formation was higher in experimental group compared to the control group (p<0.05).

Discussion

The effects of SOD and CAT supplementation to the IVM medium on oocyte maturation, cleavage and blastocyst production were assessed in our study; also the effects of addition to the IVF medium of GPx were studied by assessing the incidence of cleavage and blastocyst development. Overall, our data demonstrated a beneficial effect of addition of antioxidants to IVM and to IVF media on blastocyst growth of cat oocytes.

Endogenous and exogenous factors influence the ART outcome (Agarwal et al. 2006), one of which is OS that occurs when there is a discrepancy between ROS and antioxidant ability. ROS are involved in several physiological mechanisms including many reproductive processes (oocyte maturation, fertilization

promotion, embryo development and gestation). After fertilization, embryos can produce ROS (Guerin et al. 2001, Pero et al. 2017). Goto (1993) demonstrated that ROS production was higher in IVC in respect to in vivo mouse embryos. In the clinical ART laboratory, the risk of embryonic environmental exposure to ROS and OS is much greater than in vivo (Gough et al. 2011, Li et al. 2019). Moreover, internal stresses further increase the load of ROS that require counter-measures for con-trol. Many studies report that OS is responsible to accu-mulation of harmful metabolites that lead to follicular atresia in mammals (Devine et al. 2012). A physiologi-cal ROS amount in follicular fluid indicates a healthy developing oocyte. Conversely, a ROS increase leads to the modification of biomacromolecules damaging cells and consequently occurrence of OS. During the culture and development phases in vitro, gametes and embryos are subjected to a high amount of ROS due to deficiency of enzymatic antioxidants naturally occurring in pregnancy (Zhang et al. 2006, Takahashi 2012).

In our study, antioxidant enzymes were added to culture medium (IVM or IVF) in order to evaluated if they are able to preserve embryos from damage induced by OS. We previously demonstrated the benefi-cial effect of adding SOD to the transport medium of domestic cat ovaries (Cocchia et al. 2015b). We also demonstrated that the addition of antioxidants in the extender on refrigerated semen of fertile and infertile dogs has led to an improvement in repro-ductive performance (Del Prete et al. 2018a). Further-more, an increase in the sperm concentration and the quality of chilled semen was also achieved by supplementing the stallion diet with natural anti- oxidants (Del Prete et al. 2018b). The results showed that supplementation of IVM medium with SOD and CAT did not improve oocyte maturation rate, while it did enhance advancement to the blastocyst phase.

In vitro embryo development is highly affected by culture environment, thus in our study the purpose of adding antioxidant enzymes to IVM or IVF media was to improve in vitro conditions and decrease the detrimental effects of OS. The efficacy of SOD and CAT addition in medium for cat oocyte IVM

Table 3. Evaluation of the efficacy of GPx supplementation to in vitro fertilization (IVF) medium on embryo cleavage and blastocyst formation.

N. COCs IVC

(mean ± SD) (mean ± SD) (%)Cleaved embryo (mean ± SD) (%)N. Blastocyst

Control group 17.65 ± 4.28 5.27 ± 1.48 (29) *A 2.36 ± 0.92 (11) *A

Experimental group 18.14 ± 2.65 10.63 ± 2.15 (61) *B 4.63 ± 0.80 (28) *B

* p < 0.05 A vs B columns COCs: cumulus-oocyte complexes; IVC: in vitro culture

has not been previously reported. Recently, a study demonstrated that the supplementation in vitro with SOD and taurine in IVM and IVC media increased blastocyst development from poor-quality cat oocytes (Ochota et al. 2016).

Furthermore, we added GPx to IVF medium because, during IVF, sperm cells produce great amounts of ROS (Galecka et al. 2008). Our results showed that the rate of embryo and blastocyst formation was increased in the experimental in respect to control group.

We showed the positive effect of SOD and CAT addition to cat oocyte IVM medium and GPx in the IVF medium on in vitro feline oocyte formation. Although further studies are required to better understand the role of SOD, CAT and GPx, the data obtained encourage us to examine different concentrations of the same and/or different antioxidants, at various stages of oocyte maturation and embryo production in vitro and to evaluate possible mechanisms of action.

References

Agarwal A, Allamaneni SS (2004) Role of free radicals in female reproductive diseases and assisted reproduc-tion. Reprod Biomed Online 9: 338-347.

Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG (2006) Oxidative stress in an assisted reproductive tech-niques setting. Fertil Steril 86: 503-512.

Chi HJ, Kim JH, Ryu CS, Lee JY, Park JS, Chung DY, Choi SY, Kim MH, Chun EK, Roh SI (2008) Protective effect of antioxidant supplementation in sperm-prepara-tion medium against oxidative stress in human spermato-zoa. Hum Reprod 23: 1023-1028.

Ciani F, Cocchia N, Rizzo M, Ponzio P, Tortora G, Avallone L, Lorizio R (2008) Sex determining of cat embryo and some feline species. Zygote 16: 169-177.

Ciani F, Cocchia N, D’Angelo D, Tafuri S (2015) Influence of ROS on ovarian functions. New Discoveries in Embryology, Bin Wu ed.: 41-73.

Ciani F, Tafuri S, Troiano A, Cimmino A, Fioretto BS, Guarino AM, Pollice A, Vivo M, Evidente A, Carotenuto D, Calabrò V (2018) Anti-proliferative and pro-apoptotic effects of Uncaria tomentosa aqueous extract in squamous carcinoma cells. J Ethnopharmacol 211: 285-294.

Cocchia N, Ciani F, Russo M, El Rass R, Rosapane I, Avallone L, Tortora G, Lorizio R (2010a) Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture. Cryobiology 60: 229-234. Cocchia N, Ciani F, El-Rass R, Russo M, Borzacchiello G,

Esposito V, Montagnaro S, Avallone L, Tortora G, Lorizio R (2010b) Cryopreservation of feline epididymal sperma-tozoa from dead and alive animals and its use in assisted reproduction. Zygote 18: 1-8.

Cocchia N, Pasolini MP, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G, Mancini A (2011) Effect of SOD (superoxide dismutase) protein supplementation in semen extenders

on motility, viability, acrosome status and ERK (extracel-lular signal-regulated kinase) protein phosphoryla- tion of chilled stallion spermatozoa. Theriogenology 75: 1201-1210.

Cocchia N, Tafuri S, Abbondante L, Meomartino L, Esposito L, Ciani F (2015a) Assisted Reproductive Tech-nologies in Safeguard of Feline Endangered Species. New Discoveries in Embryology, Bin Wu ed.: 199-229. Cocchia N, Corteggio A, Altamura G, Tafuri S, Rea S,

Rosapane I, Sica A, Landolfi F, Ciani F (2015b) The effects of superoxide dismutase addition to the trans-port medium on cumulus-oocyte complex apoptosis and IVF outcome in cats (Felis catus). Reprod Biol 15: 56-64. Costantino M, Giuberti G, Caraglia A, Caraglia M,

Lombardi A, Misso G, Abbruzzese A, Ciani F, Lampa E (2009) Possible antioxidant role of SPA therapy with chlorine–sulphur–bicarbonate mineral water. Amino Acids 36: 161-165.

Del Prete C, Ciani F, Tafuri S, Pasolini MP, Della Valle G, Palumbo V, Abbondante L, Calamo A, Barbato V, Gualtieri R, Talevi R, Cocchia N (2018a) Effect of super-oxide dismutase, catalase, and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs. J Vet Sci 19: 667-675. Del Prete C, Tafuri S, Ciani F, Pasolini MP, Ciotola F,

Albarella S, Carotenuto D, Peretti V, Cocchia N (2018b) Influences of dietary supplementation with Lepidium

meyenii (Maca) on stallion sperm production and

on preservation of sperm quality during storage at 5°C. Andrology 6: 351-361.

Devine PJ, Perreault SD, Luderer U (2012) Roles of reactive oxygen species and antioxidants in ovarian toxicity. Biol Reprod 86: 27.

Esposito L, Auletta L, Ciani F, Pelagalli A, Pasolini MP, Lamagna B, Piscopo N, Amici A (2017) Hair cortisol levels in captive brown hare (Lepus europaeus): potential effect of sex, age, and breeding technology. Eur J Wildl Res 63: 62.

Formigari A, Irato P, Santon A (2007) Zinc antioxidant systems and metallothionein in metal mediated-apopto-sis: biochemical and cytochemical aspects. Comp Biochem Physiol C Toxicol Pharmacol 146: 443-459. Galecka E, Jacewicz R, Mrowicka M, Florkowski A,

Galecki P (2008) Antioxidative enzymes-structure, properties, functions. Pol Merkur Lekarski 25: 266-268. Goto Y, Noda Y, Mori T, Nakano M (1993) Increased

genera-tion of reactive oxygen species in embryos cultured

in vitro. Free Radic Biol Med 15: 69-75.

Gough DR, Cotter TG (2011) Hydrogen peroxide: a Jekyll and Hyde signalling molecule. Cell Death Dis 2: 213. Guérin P, El Mouatassim S, Ménézo Y (2001) Oxidative stress

and protection against reactive oxygen species in the pre- -implantation embryo and its surroundings. Hum Reprod Update 7: 175-189.

Hewitt DA, Watson PF, England GC (1998) Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes. Theriogenology 49: 1083-1101. Li F, Cui L, Yu D, Hao H, Liu Y, Zhao X, Pang Y, Zhu H,

Du W (2019) Exogenous glutathione improves intracellu-lar glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos. J Cell Physiol 234: 7384-7394.

Luvoni GC (2006) Gamete cryopreservation in the domestic cat. Theriogenology 66: 101-111.

Ochota M, Pasieka A, Nizanski W (2016) Superoxide dis-mutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes. Theriogenology 85: 922-927.

Pero ME, Lombardi P, Longobardi V, Boccia L, Vassalotti G, Zicarelli L, Ciani F, Gasparrini B (2017) Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilization of bovine frozen/thawed semen. Ital J Anim Sci 16: 390-392.

Pipolo S, Puglisi R, Mularoni V, Esposito V, Fuso A, Lucarelli M, Fiorenza MT, Mangia F, Boitani C (2018) Involvement of sperm acetylated histones and the nuclear isoform of glutathione peroxidase 4 in fertilization. J Cell Physiol 233: 3093-3104.

Pope CE (2014) Aspects of in vivo oocyte production, blasto-cyst development, and embryo transfer in the cat. Theriogenology 81: 126-137.

Tafuri S, Ciani F, Iorio EL, Esposito L, Cocchia N (2015) Reactive Oxygen Species (ROS) and Male Fertility. New Discoveries in Embryology, Bin Wu ed.: 19-40.

Takahashi M (2012) Oxidative stress and redox regulation on in vitro development of mammalian embryos. J Reprod Dev 58: 1-9.

Zhang M, Hong H, Zhou B, Jin S, Wang C, Fu M, Wang S, Xia G (2006) The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa. J Endocrinol 189: 493-507.